CN107383927B - A kind of nitrine indoles dimethime Molecule of Cyanine Dyes and application - Google Patents
A kind of nitrine indoles dimethime Molecule of Cyanine Dyes and application Download PDFInfo
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- CN107383927B CN107383927B CN201710447796.9A CN201710447796A CN107383927B CN 107383927 B CN107383927 B CN 107383927B CN 201710447796 A CN201710447796 A CN 201710447796A CN 107383927 B CN107383927 B CN 107383927B
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- 239000000975 dye Substances 0.000 title claims abstract description 28
- -1 nitrine indoles Chemical class 0.000 title claims abstract description 21
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 title claims abstract 8
- 241000894006 Bacteria Species 0.000 claims abstract description 69
- 238000001514 detection method Methods 0.000 claims abstract description 8
- 238000004043 dyeing Methods 0.000 claims abstract description 6
- 238000006243 chemical reaction Methods 0.000 abstract description 19
- 210000004027 cell Anatomy 0.000 abstract description 17
- 210000000170 cell membrane Anatomy 0.000 abstract description 10
- 230000003321 amplification Effects 0.000 abstract description 8
- 238000003199 nucleic acid amplification method Methods 0.000 abstract description 8
- 230000001580 bacterial effect Effects 0.000 abstract description 6
- 230000035699 permeability Effects 0.000 abstract description 4
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 abstract description 3
- 230000002255 enzymatic effect Effects 0.000 abstract description 2
- 150000002475 indoles Chemical class 0.000 abstract 2
- 150000002596 lactones Chemical class 0.000 abstract 1
- 125000004433 nitrogen atom Chemical group N* 0.000 abstract 1
- 238000002331 protein detection Methods 0.000 abstract 1
- 230000004083 survival effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 24
- 150000001875 compounds Chemical class 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 20
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 16
- 241000191967 Staphylococcus aureus Species 0.000 description 16
- 241000607142 Salmonella Species 0.000 description 14
- 241001333951 Escherichia coli O157 Species 0.000 description 11
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 239000007788 liquid Substances 0.000 description 9
- 238000002360 preparation method Methods 0.000 description 9
- 229910001868 water Inorganic materials 0.000 description 9
- QGKMIGUHVLGJBR-UHFFFAOYSA-M (4z)-1-(3-methylbutyl)-4-[[1-(3-methylbutyl)quinolin-1-ium-4-yl]methylidene]quinoline;iodide Chemical compound [I-].C12=CC=CC=C2N(CCC(C)C)C=CC1=CC1=CC=[N+](CCC(C)C)C2=CC=CC=C12 QGKMIGUHVLGJBR-UHFFFAOYSA-M 0.000 description 8
- 235000019441 ethanol Nutrition 0.000 description 8
- 239000007787 solid Substances 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000371 Esterases Proteins 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 238000004128 high performance liquid chromatography Methods 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 229940126214 compound 3 Drugs 0.000 description 6
- 235000013305 food Nutrition 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 239000011541 reaction mixture Substances 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 229940125898 compound 5 Drugs 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 238000006731 degradation reaction Methods 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000005286 illumination Methods 0.000 description 4
- 150000007523 nucleic acids Chemical class 0.000 description 4
- 102000039446 nucleic acids Human genes 0.000 description 4
- 108020004707 nucleic acids Proteins 0.000 description 4
- 239000008223 sterile water Substances 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000004367 Lipase Substances 0.000 description 3
- 108090001060 Lipase Proteins 0.000 description 3
- 102000004882 Lipase Human genes 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 229940125904 compound 1 Drugs 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 3
- WBJINCZRORDGAQ-UHFFFAOYSA-N ethyl formate Chemical compound CCOC=O WBJINCZRORDGAQ-UHFFFAOYSA-N 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 229910052736 halogen Inorganic materials 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000002401 inhibitory effect Effects 0.000 description 3
- 235000019421 lipase Nutrition 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 230000000149 penetrating effect Effects 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000010992 reflux Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 229910052721 tungsten Inorganic materials 0.000 description 3
- 239000010937 tungsten Substances 0.000 description 3
- 239000012224 working solution Substances 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- 238000007400 DNA extraction Methods 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 230000004087 circulation Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000007071 enzymatic hydrolysis Effects 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 230000008020 evaporation Effects 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 238000009533 lab test Methods 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 150000003053 piperidines Chemical class 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- WSGYTJNNHPZFKR-UHFFFAOYSA-N 3-hydroxypropanenitrile Chemical compound OCCC#N WSGYTJNNHPZFKR-UHFFFAOYSA-N 0.000 description 1
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 241001646719 Escherichia coli O157:H7 Species 0.000 description 1
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical compound ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- 239000007832 Na2SO4 Substances 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical group OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 241000607768 Shigella Species 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000012267 brine Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 235000013365 dairy product Nutrition 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000009881 electrostatic interaction Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000002979 esterase activity determination Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 125000001041 indolyl group Chemical group 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 230000009149 molecular binding Effects 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 239000012044 organic layer Substances 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 1
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical compound O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
- C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
- C09B23/10—The polymethine chain containing an even number of >CH- groups
- C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- Bioinformatics & Cheminformatics (AREA)
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- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Biomedical Technology (AREA)
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of nitrine indoles dimethime Molecule of Cyanine Dyes and application, nitrine indoles dimethime cyanine dyes of the invention introduces the side chain containing ester bond and azido group using indoles dimethime cyanine dyes as core skeleton on the nitrogen-atoms of indoles.Nitrine indoles dimethime cyanine dyes of the present invention has permeability of cell membrane, it can be used for protein detection, living cells dyeing, and the survival condition of bacterium is judged based on bacterial lactone enzymatic activity, the amplification of the dead bacterium of selective depression in the reaction, to achieve the purpose that bacterium rapid quantitative detection living.Dye molecule of the present invention has more application prospect in terms of living stems.
Description
Technical field
The present invention relates to biological field, be related to it is a kind of suitable for cell dyeing, the molecule of living stems structure and its
Application in Bacteria Detection technology.
Background technique
WHO report shows global 40~6,000,000,000 food origin diseases of generation every year, wherein about 70% is because of biogenic dirt
It contaminates caused by food.The important pathogenic bacteria recognized in the world that can cause food origin disease, is such as widely present in dairy produce, vegetable
Salmonella, staphylococcus aureus, Listeria monocytogenes, colon bacillus 0157, will in dish, aquatic products, meat products etc. are congratulated
Salmonella etc. is an important factor for influencing food safety.Microorganism exists with a variety of physiological status: educable viable bacteria, in non-
Can cultivation conditions viable bacteria (Viable but Non-culturable, VBNC), with complete structure but inactive it is dead
Bacterium (" Ghosts ") and the dead bacterium of membrane damage.Only " viable bacteria " just remains with the virulence of opportunistic pathogen and pathogenic, food-safe to make
It is threatened at potential, and the bacterium Jing Guo the various approach inactivation treatments such as high temperature, ultraviolet light has lost that its is pathogenic, therefore food
The key that borne pathogen is examined is that the pathogenic bacteria of " work " in food whether there is and accurate quantitative analysis.
Standard currently used for difference viable bacteria and dead bacterium mainly has whether can cultivate, if has metabolic activity and cell membrane
It is whether complete.Isolated culture will appear missing inspection for the bacterium of viable but non-culturable state.Viable bacteria EMA/PMA-PCR detects skill
Art is a kind of high specific rapidly growing in recent years, highly sensitive nucleic acid detection method.Detection according to be EMA and PMA all
There is no permeability of cell membrane, EMA and PMA can only penetrate into the cell of membrane damage, covalent bond occur with DNA, so that film be inhibited to damage
Hurt the amplification of DNA in the reaction in cell.There is studies have shown that (to be such as present in some reversible part film damaging cells
Bacterium in environmental samples), dyestuff causes different degrees of viable bacteria DNA to lose it is possible to penetrating cell film, generates false negative
As a result.In addition, being not directly placed on the sterilizing methods of cell membrane for ultraviolet sterilization, cold sterilization etc., can not adopt
With the effect for assessing sterilizing methods based on the viable bacteria PCR method of EMA or PMA, it otherwise will cause viable count purpose and over-evaluate.Institute
With, cell membrane damage may be a kind of extreme performance form of bacterial death, using the integrality of cell membrane as distinguish viable bacteria and
The examination criteria of dead bacterium is still defective, it is therefore desirable to propose a kind of more scientific differentiation standard.
Summary of the invention
The nitrine indoles dimethime cyanines dye based on bacterial esterase Activity determination number of viable that it is an object of the present invention to provide a kind of
Expect molecule (abbreviation IDA) and applies.The nitrine indoles dimethime Molecule of Cyanine Dyes has permeability of cell membrane, not only can be with
It into dead cell, can also pass into living cells, after which enters cell, bacterial esterase can be with water in living cells
For ester bond on solution dye molecule to make azido group fall off, esterase is inactive in dead cell causes azido group that can not fall off
And crosslink to inhibit the amplification of DNA in dead cell with DNA molecular, achieve the purpose that detect viable bacteria.
The object of the invention is achieved through the following technical solutions:
A kind of nitrine indoles dimethime Molecule of Cyanine Dyes, the molecular structure include: conjugated group, enzyme mark group, crosslinking group
Group and four part of connecting portion.Molecular structure is as follows:
Rapid fluorescence dyeing and viable bacteria content detection of the above-mentioned nitrine indoles dimethime Molecule of Cyanine Dyes for cell.
Nitrine indoles dimethime Molecule of Cyanine Dyes synthetic method of the present invention is simple, and raw material is cheap and easy to get, and synthetic route is as follows:
Compound 3 is reacted to obtain by compound 1 with bromo-acetic acid tert-butyl；Tetrahydrofuran dissolved compound 1 is first used, is then existed
At 0 DEG C be added NaH after stirring 30min bromo-acetic acid tert-butyl is added dropwise again, after being extracted with ethyl acetate, by drying, filtering,
Compound 3 can be obtained after evaporation.
Compound 6 is reacted to obtain by compound 3 with compound 5；Compound 5 is by being added CH after ethyl alcohol dissolved compound 43I
It is concentrated after being stirred overnight at 85 DEG C, obtains after purification.A drop piperidines, reaction is added with ethyl alcohol dissolved compound 3 and compound 5
Object obtains compound 6 after being refluxed overnight.
Compound 7 is added concentrated hydrochloric acid by compound 6 and is stirred overnight reflux, after LCMS shows fully reacting, by filtering, washing
Wash, dry after obtain.
Compound 8 is anti-at room temperature by compound 7 and ethanedioly chloride under the catalysis of methylene chloride and dimethylformamide
It should obtain.
Target compound is added compound 8 and 2- nitrine ethyl alcohol is stirred at room temperature using methylene chloride as solvent
Night obtains after being filtered, washed, drying, and target compound is red solid.
Nitrine indoles dimethime Molecule of Cyanine Dyes structure of the present invention passes through connecting portion for crosslinked group, enzyme mark base
Group and conjugated group connect, formed it is a kind of can free penetrating cell membrane, it is sensitive to esterase active, divide with crosslinked action
Son.The effect of conjugated group is to make molecule can be with free penetrating cell membrane, therefore molecule is able to enter all cells.It is of the present invention
The conjugated group of molecule is indoles dimethime phthalocyanine molecule, is good DNA fluorescence probe.One end of ester bond in enzyme mark group with
Crosslinked group is connected, and the other end is connected by connecting portion and conjugated group.Since the ester bond in enzyme mark group is to biology
Esterase active is sensitive, and the ester linkage breaking under esterase hydrolyzed effect, conjugated group can be separated from each other with crosslinked group.This is also this hair
The key component of the bright molecular structure being related to.Crosslinked group under light illumination with can inhibit nucleic acid in the reaction after nucleic acid covalent bond
Amplification.In competent cell, when the ester bond in molecule is broken by enzymatic hydrolysis, crosslinked group falls off from molecule；Conversely, in nothing
Active intracellular, crosslinked group will not fall off, and under the action of visible light and covalently covalent compound is cross-linked to form, after inhibition
Continuous amplification.And the molecule of extra free state, crosslinked group because reacted under visible light action with water generate azanol due to by
Passivation.Therefore, it will be joined together with molecule provided by the invention with quantitative technique, and establish novel living stems technology to distinguish
Living, dead bacterium will depend on the intracorporal esterase active of cell, rather than cell membrane integrity will be than previous living stems skill
Art is more acurrate.
Compared with the prior art, the present invention has the following advantages that and technical effect:
(1) under the premise of not influencing molecular binding group function, the conjugated group of dyestuff of the present invention is dimethime cyanines dye
Material, the phosphoric acid backbone in electropositive and DNA double helical structure which passes through itself generate electrostatic interaction, make dyestuff
The freedom degree of methine chain rotation reduces, and causes the fluorescence intensity of dyestuff to enhance, the dyeing effect under fluorescence microscope is more preferable.
(2) experiments have shown that, IDA molecule can effectively penetrate bacteria wall film, and have good binding ability with nucleic acid, with fat
IDA is hydrolyzed in enzyme, and percent hydrolysis is up to 96%.
(3) conjugated group of the present invention increases a phenyl ring, enhances the rigidity of dye molecule, experiments have shown that, in PCR mistake
Cheng Zhongneng preferably inhibits the amplification of dead bacterium DNA, and the dead bacterium DNA handled through IDA molecule is than the dead bacterium that handles without IDA molecule
The increased Ct value of DNA is up to 16, and influence of the present invention to viable bacteria DNA cloning is smaller, and the viable bacteria DNA ratio handled through IDA molecule is not
The Ct value of the viable bacteria DNA handled through IDA molecule is differed down to 0.6.As it can be seen that the IDA molecule after improvement, not only remains original point
Sub- key function characteristic, esterase degradation rate is higher, can preferably inhibit the expansion of dead bacterium on the basis of influencing very little to viable bacteria
Increase, it is more acurrate to can be applied to living stems result.
(4) the improved IDA molecule of molecular structure is analyzed from synthetic technology, and connecting portion alkyl length is C1, can be big
The big molecule that reduces generates the probability of isomer in the synthesis process, to improve yield, reduces in preparation process because of same point
It is lost caused by isomers.
Detailed description of the invention
Fig. 1 is the chemical structural drawing for marking the nitrine indoles dimethime Molecule of Cyanine Dyes of each radical position.
Fig. 2 is the shows fluorescent microscopy images mixed with the IDA molecule of 10 μ g/mL concentration with staphylococcus aureus after being incubated for
Picture.
Fig. 3 is the degradation rate of IDA molecule with the figure of changing of fatty enzyme dosage.
Fig. 4 is that the IDA molecule of various concentration respectively expands the DNA of the work of three kinds of bacterium, dead bacterium mixed bacteria liquid in PCR reaction
The effect picture of increasing from left to right respectively represents staphylococcus aureus, Escherichia coli O 157 and salmonella.
Fig. 5 is by IDA and without staphylococcus aureus, Escherichia coli O 157 and the Salmonella by IDA processing
The PCR reaction result figure of bacterium viable bacteria DNA.
Fig. 6 is by IDA and without staphylococcus aureus, Escherichia coli O 157 and the Salmonella by IDA processing
The PCR reaction result figure of the dead bacterium DNA of bacterium.
Fig. 7 is by IDA and without staphylococcus aureus, Escherichia coli O 157 and the Salmonella by IDA processing
The PCR reaction result figure of bacterium Mixed Microbes DNA living, dead.
Specific embodiment
The present invention will be further described with reference to the examples below, however, the present invention is not limited thereto.In the following example not
The experimental method of actual conditions is indicated, usually according to normal condition, such as filtering, rotary evaporation, purifying column purification.
Embodiment 1
A kind of nitrine indoles dimethime Molecule of Cyanine Dyes (IDA), preparation method is as follows:
Specific preparation process is as follows:
The preparation of compound 3:
0.3g NaH is added after being dissolved with 10mL tetrahydrofuran in 1.0g compound 1, and 30min is stirred at 0 DEG C, continues 0
1.48g bromo-acetic acid tert-butyl is added dropwise at DEG C, reaction mixture temperature stirs 1h after being warmed to room temperature.Thin-layered chromatography is shown instead
After answering completely, it is extracted with ethyl acetate after salt water quenching reaction is added.It is washed with brine organic layer, uses Na2SO4After drying, mistake
Filter, evaporation obtain thick material.Lurid solid, yield are obtained after thick material is dry after being washed with PE (phosphate buffer)
67%.
The preparation of compound 5:
1.0g compound 4 is dissolved in seal pipe with 100mL ethyl alcohol, and 7.1g MeI is added, stirs at 85 DEG C after mixing
Overnight.After liquid chromatography shows fully reacting, with silica gel column chromatography (DCM/MeOH=10/1~CAN acetonitrile/H2O=10/
1) reaction mixture is concentrated, obtains 5.0g light yellow solid, yield 51% after purification.The structure of compound passes through efficient liquid phase
Chromatography characterization.
LCMS:(M+1) +=164.2, Rt=2.5min.HPLC:95%, Rt=4.777min.
The preparation of compound 6:
1.0g compound 3 and the 50mL ethyl alcohol of 1.0g compound 5 dissolve, and 1 drop piperidines catalysis is added.Mixture is stirred overnight
Reflux.After liquid chromatography shows fully reacting, reaction mixture is filtered, it is solid with 1.3g red is dried to obtain after ethanol washing
Body, yield 71%.The structure of compound is characterized by high performance liquid chromatography.
LCMS:(M) +=405.2, Rt=3.9min.HPLC:97%, Rt=5.215min.
The preparation of compound 7:
1.3g compound 6 is made suspension with 100mL concentrated hydrochloric acid and is stirred overnight reflux.Liquid chromatography shows fully reacting
Afterwards, reaction mixture is filtered, with being dried to obtain 1.1g red solid, yield 94% after ethanol washing.The structure of compound passes through
Cross high performance liquid chromatography characterization.
LCMS:(M+1) +=349.2, Rt=3.4min.
The preparation of compound 8:
12mL ethanedioly chloride is added after being dissolved with 50mL methylene chloride in 150mg compound 7, adds a drop dimethyl formyl
Amine is stirred at room temperature overnight as catalyst, mixture.It, will after liquid chromatography display fully reacting (methanol quenching reaction)
Red solid is obtained after reaction mixture concentration directly synthesizes final product with next step.
The preparation of target compound:
4mL 2- azidoethyl alcohol is added after being dissolved with 240mL methylene chloride in 150mg compound 8.Reaction mixture is in room
It is stirred overnight under temperature.After liquid chromatography shows fully reacting, filtering is dried to obtain red solid after being washed with methylene chloride,
Yield 58%.
IDA molecular structure is as shown in Figure 1.Its relative molecular mass is 418.2.It is red solid under IDA room temperature, is slightly soluble in
Water, cannot be complete molten in methylene chloride, ethyl acetate, is soluble in dimethyl sulfoxide, unstable in methanol acetonitrile.Its liquid phase color
It is as follows to compose qualification result: LCMS:(M+1) +=418.2, Rt=3.6min.HPLC:96%, Rt=4.82min.1H NMR
(400MHz, DMSO-d6) δ 8.51-8.44 (d, J=15.6Hz, 1H), 8.43 (s, 1H), 8.36 (d, J=7.4Hz, 1H),
8.31 (m, 1H), 8.18 (d, J=8.4Hz, 1H), 7.86-7.77 (m, 1H), 7.71 (m, 1H), 7.64 (m, 1H), 7.57 (d, J
=15.6Hz, 1H), 7.43-7.35 (m, 2H), 5.43 (s, 2H), 4.31 (m, 5H), 3.66-3.59 (m, 1H).
Embodiment 2
The permeability test of bacterial membrane is carried out to the dyestuff that embodiment 1 obtains.Take 500 μ L staphylococcus aureus bacterium outstanding
For liquid in 1.5mL centrifuge tube, 4000g is centrifuged 5min, and isometric sterile water is resuspended.The 0.5mg/ dissolved with DMSO is added in bacterium solution
The 1 μ L of IDA working solution of mL makes the final concentration of 10 μ g/mL of IDA in bacterium solution, and after mixing well, dark place is incubated for 10min at room temperature.
Centrifuge tube is placed horizontally on ice, 5min illumination is carried out at the tungsten halogen lamp lower section 20cm of 650W, during which jiggles ice chest
Guarantee that solution is sufficiently irradiated.Negative control group replaces IDA solution using sterile ultrapure water.
It is fixed to through IDA treated thallus using 10% formalin buffer.Draw the fixed bacterium finished of 5 μ L
Liquid is dripped in the glass slide center of no autofluorescence, glycerol mountant is added, is closed after covered using transparent nail polish.
It is made after sample with confocal laser scanning microscope result (excitation wavelength 488nm, launch wavelength 620nm, 100 times of oil mirrors).
Through IDA, treated that thallus is colored, and issues intense fluorescence under excitation light, the dyeing part of dyestuff is mainly
Core area only has faint coloring to cytoplasm, this illustrates that dyestuff can pass through living cells film and carry out selectively staining to DNA, as a result
As shown in Figure 2.
Embodiment 3
The sensibility of IDA of the invention to lipase:
Enzymatic treatment condition: by 0.105g/L IDA solution 5mL (2.5 μm of oL), 2min, lipase are preheated in 40 DEG C of water-baths
0.5~3.5U of dosage after mixing well, handles 20min under 40 DEG C, 150r/min dark.Membrane filtration, filtrate is for efficient
Product (being protected from light) after the hydrolysis of liquid chromatogram (HPLC) analysis detection.HPLC condition: chromatographic column uses Zorbax SB-C18 column
(4.6mm × 250mm, 5 μm)；Mobile phase is water: phosphate buffer (0.02mol/L, the pH of acetonitrile=60:40 (volume ratio)
Value 6.99~7.01)；30 DEG C of column temperature；Flow 1mL/min；10 μ L of sample volume；Using PDA detector, excitation wavelength 488nm, hair
The long 520nm of ejected wave.In control group, enzymolysis liquid is replaced without the IDA solution of enzymatic hydrolysis using isoconcentration.As a result it is indicated with degradation rate,
Calculation formula is as follows: degradation rate=(IDA content after IDA content-enzymatic hydrolysis before digesting)/control group IDA content × 100%.
For IDA molecule under lipase-catalyzed, percent hydrolysis is up to 96%, illustrates that IDA molecule is sensitive to lipase active, energy
The fast hydrolyzing under corresponding enzyme effect, as a result as shown in Figure 3.
Embodiment 4
Inhibiting effect of the IDA to DNA cloning
500 are taken to take 500 μ L staphylococcus aureuses, Escherichia coli O 157: H7, Shigella and single increasing Lee respectively respectively
This special bacterium bacterium solution is separately added into 0.5,1,3,5,10,15,20 μ L of IDA working solution in 1.5mL centrifuge tube, in bacterium solution to be made in bacterium solution
IDA final concentration is respectively 0.5,1,3,5,10,15,20 μ g/mL, and after mixing well, dark place is incubated for 10min at room temperature.It will be from
Heart pipe is placed horizontally on ice, and carrying out 5min illumination at the tungsten halogen lamp lower section 20cm of 650W is crosslinked IDA and DNA, while blunt
Change the IDA molecule to dissociate in solution, during which jiggles ice chest and guarantee that solution is sufficiently irradiated.It will be by the molten of lighting process
Liquid investigates IDA molecule to the inhibiting effect of DNA cloning as real time fluorescent quantitative reaction template.
Quantitative fluorescent PCR reaction system total volume is 20 μ L, wherein including 2 × Taq PCR MasterMix10 μ L, ROX
0.2 μ L, each 1 μ L of front and back primer (10 μm of ol/L), probe (10 μm of ol/L) 0.5 μ L, 5.3 μ L of DEPC water, 2 μ L of DNA profiling solution.
Reaction condition: then 95 DEG C of initial denaturations 2min, 95 DEG C of denaturation 5s are cooled to 60 DEG C and keep 40s (collecting fluorescence signal), the mistake
Cheng Jinhang 40 circulations.40 DEG C of heat preservation 2min after reaction.Each quantitative fluorescent PCR reacts 3 parallel laboratory tests of each progress, meter
Calculate Average Ct values (the threshold value recurring number experienced that fluorescence signal reaches setting) and sample standard deviation SD value.
The IDA that concentration is 10 μ g/mL has apparent inhibiting effect to the reaction amplification of three kinds of bacteriums, as a result such as Fig. 4 institute
Show.
Embodiment 5
IDA detects staphylococcus aureus, Escherichia coli O 157 and salmonella viable bacteria
Staphylococcus aureus, Escherichia coli O 157 and the salmonella bacterium of the fresh cultured of two parts of isoconcentrations are taken respectively
In centrifuge tube, sterile water washing thalline is resuspended after twice with isometric sterile water liquid 1.5mL.A copy of it bacterium solution is placed in
5min is heated in 100 DEG C of boiling water baths and carries out hot inactivation, obtains dead bacterium bacterium solution.Viable bacteria bacterium solution, dead bacterium bacterium solution and dead, work are taken respectively
Bacterium ratio is each two parts of Mixed Microbes bacterium solution of 1:1, and IDA working solution, which is added, in a copy of it into every kind of bacterium solution makes IDA in bacterium solution
Final concentration of 10 μ g/mL, another does negative control with sterile water.After mixing well, it is protected from light incubation 10 minutes at room temperature.It will
Centrifuge tube is placed horizontally on ice, carries out 10min illumination below the tungsten halogen lamp of 650W at 20cm, during which jiggles ice chest guarantor
Card solution is sufficiently irradiated.It will be handled by IDA and be centrifuged 5min without the bacterium solution 4000g that IDA is handled, remove supernatant.Make
DNA is extracted with bacterial genomes extracts kit, the DNA after extraction carries out PCR reaction as template.
Quantitative fluorescent PCR reaction system total volume is 20 μ L, wherein including 2 × Taq PCR MasterMix10 μ L, ROX
0.2 μ L, each 1 μ L of front and back primer (10 μm of ol/L), probe (10 μm of ol/L) 0.5 μ L, 5.3 μ L of DEPC water, 2 μ L of DNA profiling solution.
Reaction condition: then 95 DEG C of initial denaturations 2min, 95 DEG C of denaturation 5s are cooled to 60 DEG C and keep 40s (collecting fluorescence signal), the mistake
Cheng Jinhang 40 circulations.40 DEG C of heat preservation 2min after reaction.Each quantitative fluorescent PCR reacts 3 parallel laboratory tests of each progress, meter
Calculate Average Ct values (the threshold value recurring number experienced that fluorescence signal reaches setting) and sample standard deviation SD value.To experimental result into
Row is drawn, and abscissa represents IDA concentration, and ordinate handles to obtain with the Ct value-handled through IDA molecule without IDA molecule
Ct value, △ Ct indicates.
The PCR result that IDA detects staphylococcus aureus, Escherichia coli O 157 and salmonella viable bacteria is as shown in Figure 5.
The result shows that by IDA handle than handle without IDA staphylococcus aureus, Escherichia coli O 157 and
For the Ct value difference of salmonella viable bacteria DNA down to 0.6, this illustrates that influence of the IDA processing to viable bacteria quantitative PCR detection is little.
Embodiment 6
Staphylococcus aureus, the Escherichia coli that will be handled through the IDA of 10 μ g/mL of over-richness and handled without IDA
After O157 and the dead bacterium of salmonella carry out DNA extraction, the DNA of purifying is reacted as template respectively, as a result such as Fig. 6 institute
Show.
The result shows that by IDA handle than handle without IDA staphylococcus aureus, Escherichia coli O 157 and
The Ct value of the dead bacterium DNA of salmonella is high by 16 or so, this can inhibit the amplification of dead bacterium DNA after illustrating IDA processing well.
Embodiment 7
By the IDA processing by 10 μ g/mL and the staphylococcus aureus handled without IDA, Escherichia coli O 157
And after the work of salmonella, dead bacterium mixed bacteria liquid carry out DNA extraction, respectively react the DNA of purifying as template, it ties
Fruit is as shown in Figure 7.
The result shows that by IDA handle than handle without IDA staphylococcus aureus, Escherichia coli O 157 and
The work of salmonella, the Ct value of dead bacterium mixed bacteria liquid are high by 10 or so, illustrate the shadow that dead bacterium background can be excluded after IDA is handled
It rings, detects that number of viable achievees the purpose that detect viable bacteria.
Claims (3)
1. a kind of nitrine indoles dimethime Molecule of Cyanine Dyes, which is characterized in that molecular structure is as follows:
2. the application of nitrine indoles dimethime Molecule of Cyanine Dyes described in claim 1, which is characterized in that the molecule is used for viable bacteria
The detection of content.
3. the application of nitrine indoles dimethime Molecule of Cyanine Dyes described in claim 1, which is characterized in that the molecule is used for thin
Born of the same parents carry out rapid fluorescence dyeing.
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| JPH06110156A (en) * | 1992-09-25 | 1994-04-22 | Konica Corp | Silver halide photographic sensitive material |
| CN104194379A (en) * | 2014-08-08 | 2014-12-10 | 华南理工大学 | Thiazole orange cyanine dye molecule and application thereof |
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| 吲哚二甲川菁的合成、晶体结构、光谱性质及生物应用;张江华等;《高等学校化学学报》;20151010;第36卷(第10期);1924-1932 |
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